NMR Analysis Suggests Synergy between the RRM2 and the Carboxy-Terminal Segment of Human La Protein in the Recognition and Interaction with HCV IRES.

The La protein (lupus antigen) is a ubiquitous RNA-binding protein found in all human cells. It is mainly localized in the nucleus, associates with all RNA polymerase III (Pol III) transcripts, as the first factor they interact with, and modulates subsequent processing events. Export of La to the cytoplasm has been reported to stimulate the decoding of specific cellular and viral mRNAs through IRES-dependent (Internal ribosome entry site) binding and translation. Using NMR (Nuclear Magnetic Resonance) spectroscopy, we provide atomic-level-resolution structural insights on the dynamical properties of human La (hLa) protein in solution. Moreover, using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we provide evidence about the role and ligand specificity of the C-terminal domain of the La protein (RRM2 and C-terminal region) that could mediate the recognition of HCV-IRES.

Mapping of the sGC Stimulator BAY 41-2272 Binding Site on H-NOX Domain and Its Regulation by the Redox State of the Heme.

Soluble guanylate cyclase (sGC) is the main receptor of nitric oxide (NO) and by converting GTP to cGMP regulates numerous biological processes. The ß1 subunit of the most abundant, α1ß1 heterodimer, harbors an N-terminal domain called H-NOX, responsible for heme and NO binding and thus sGC activation. Dysfunction of the NO/sGC/cGMP axis is causally associated with pathological states such as heart failure and pulmonary hypertension. Enhancement of sGC enzymatic function can be effected by a class of drugs called sGC "stimulators," which depend on reduced heme and synergize with low NO concentrations. Until recently, our knowledge about the binding mode of stimulators relied on low resolution cryo-EM structures of human sGC in complex with known stimulators, while information about the mode of synergy with NO is still limited. Herein, we couple NMR spectroscopy using the H-NOX domain of the Nostoc sp. cyanobacterium with cGMP determinations in aortic smooth muscle cells (A7r5) to study the impact of the redox state of the heme on the binding of the sGC stimulator BAY 41-2272 to the Ns H-NOX domain and on the catalytic function of the sGC. BAY 41-2272 binds on the surface of H-NOX with low affinity and this binding is enhanced by low NO concentrations. Subsequent titration of the heme oxidant ODQ, fails to modify the conformation of H-NOX or elicit loss of the heme, despite its oxidation. Treatment of A7r5 cells with ODQ following the addition of BAY 41-2272 and an NO donor can still inhibit cGMP synthesis. Overall, we describe an analysis in real time of the interaction of the sGC stimulator, BAY 41-2272, with the Ns H-NOX, map the amino acids that mediate this interaction and provide evidence to explain the characteristic synergy of BAY 41-2272 with NO. We also propose that ODQ can still oxidize the heme in the H-NOX/NO complex and inhibit sGC activity, even though the heme remains associated with H-NOX. These data provide a more-in-depth understanding of the molecular mode of action of sGC stimulators and can lead to an optimized design and development of novel sGC agonists.

Replacement of heme by soluble guanylate cyclase (sGC) activators abolishes heme-nitric oxide/oxygen (H-NOX) domain structural plasticity.

The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a ß1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC "activators" aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules.

Backbone and side chain NMR assignments of the H-NOX domain from Nostoc sp. in complex with BAY58-2667 (cinaciguat).

Soluble guanylate cyclase (sGC) enzyme is activated by the gaseous signaling agent nitric oxide (NO) and triggers the conversion of GTP (guanosine 5'-triphosphate) to cGMP (cyclic guanylyl monophosphate). It contains the heme binding H-NOX (heme-nitric oxide/oxygen binding) domain which serves as the sensor of NO and it is highly conserved across eukaryotes and bacteria as well. Many research studies focus on the synthesis of chemical compounds bearing possible therapeutic action, which mimic the heme moiety and activate the sGC enzyme. In this study, we report a preliminary solution NMR (Nuclear Magnetic Resonance) study of the H-NOX domain from Nostoc sp. cyanobacterium in complex with the chemical sGC activator cinaciguat (BAY58-2667). An almost complete sequence-specific assignment of its 1H, 15N and 13C resonances was obtained and its secondary structure predicted by TALOS+.

Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops ß5-α3 and α3-ß6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated "dark" conformational state.

Therapeutic Targeting of the Soluble Guanylate Cyclase.

The soluble guanylate cyclase (sGC) is the physiological sensor for nitric oxide and alterations of its function are actively implicated in a wide variety of pathophysiological conditions. Intense research efforts over the past 20 years have provided significant information on its regulation, culminating in the rational development of approved drugs or investigational lead molecules, which target and interact with sGC through novel mechanisms. However, there are numerous questions that remain unanswered. Ongoing investigations, with the critical aid of structural chemistry studies, try to further elucidate the enzyme's structural characteristics that define the association of "stimulators" or "activators" of sGC in the presence or absence of the heme moiety, respectively, as well as the precise conformational attributes that will allow the design of more innovative and effective drugs. This review relates the progress achieved, particularly in the past 10 years, in understanding the function of this enzyme, and focusses on a) the rationale and results of its therapeutic targeting in disease situations, depending on the state of enzyme (oxidized or not, heme-carrying or not) and b) the most recent structural studies, which should permit improved design of future therapeutic molecules that aim to directly upregulate the activity of sGC.

NMR study of non-structural proteins--part II: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV).

Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/ß-fold.